Tris-maleimido compounds as intermediates in trifunctional antibody synthesis

ABSTRACT

The present invention is directed to a compound for use as an intermediate in the production of novel trifunctional antibody-like compounds. More particularly, the present invention is directed to a compound of the formula: ##STR1## wherein X is ##STR2## wherein k=1 or 0; 
     wherein Z is ##STR3## wherein s=1 or 0; 
     wherein n=1 or 0; 
     wherein q=1 or 0; 
     wherein Y is ##STR4## wherein Y&#39;, is ##STR5## wherein p or m may be the same or different and are integers ranging from 0 to 20 with the proviso that when n=0, the sum of m and p is an integer ranging from 1 to 20, whereas when n=1, p and m are each an integer that is at least 1 and the sum of p and m is an integer ranging from 2 to 20; 
     wherein R 1  is straight or branched chain lower alkyl having from 1 to 6 carbon atoms or lower alkoxy having from 1-6 carbon atoms; and 
     wherein R 2  is hydrogen, phenyl, --COOH, or straight or branched chain lower alkyl having from 1-6 carbon atoms, with the proviso that the lower alkyl moiety may be mono substituted by --NH 2 , --OH, or --COOH. 
     The compound of the present invention is useful as a trivalent coupling agent for linking Fab&#39;-like fragments to form both bifunctional and trifunctional antibody-like compounds.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention is directed to a trivalent coupling agent. Moreparticularly, the present invention is directed to a tris-maleimidocompound having three linker arms of variable length, charge, labilityand hydrophobicity. Such compounds are useful as intermediates in thesynthesis of bifunctional and trifunctional (i.e., tri.specific)antibody-like compounds which are useful in medical diagnoses,therapeutics and diagnostic/therapeutic combinations.

B. Background

Antibodies are complex protein molecules generated by an organism'simmune system in response to an antigen perceived by the host as beingforeign. The extreme plasticity and diversity of an animal's immunerepertoire permits the generation of an enormous variety of antibodymolecules to an equally large number of antigens. However, individualantibodies are monospecific and therefore merely monofunctional forpurposes of this invention.

Landsdorp, et al. teaches the formation of a bifunctional antibodycomplex formed by cross linking two monoclonal antibodies of differentspecificities but of the same isotype. Landsdorp, et al., "CyclicTetramolecular Complexes Of Monoclonal Antibodies: A New Type OfCross-linking Agent," Eur. J. Immunol., 16, p. 679-83 (1986).Landsdorp's cross-linking agent consisted of two anti-isotype antibodymolecules, which cross-linked the two monoclonal antibodies to form acyclic tetramolecular complex that was bifunctional. Implicitly, theintact antibodies taught by Landsdorp have Fc regions which are capableof binding complement and/or stimulating an immune response if presentedin vivo.

An object of the present invention is to avoid the use of antibodies orlinkers having Fc regions which may bind, complement, and/or stimulatean immune response by the antibody target complex.

Reading U.S. Pat. No. 4,714,681 teaches the creation of bifunctionalantibody-antibody chimeras by the fusion of two different hybridoma celllines which produce monoclonal antibodies of different specificities(quadroma) and by the fusion of a hybridoma producing a specificmonoclonal antibody with a lymphocyte producing a different antibody.The success of this method depends on the ability of the hybrid cells toproduce both the heavy and light chains of both parental types in equalamounts such as to maximize the potential for the random assembly ofheavy and light chains to yield the appropriate bifunctional complex. Atbest, this random assembly of antibody subunits can result in only oneof eight molecules (12.5%) being of the desired specificities. Moreover,Reading's chimeras are whole antibodies that have intact Fc regions.Consequently, when injected into a "foreign" species, Reading'schimeras, like Langsdorp's bifunctionals, have the potential to invokean interaction with components of the immune system that bear Fcreceptors (e.g., macrophages, complement, etc.).

Among the first descriptions of the use of chemical compounds tocovalently cross-link antibodies was Hamaguchi et al., J. Biochem. 85;1289-1300 (1979). Hamaguchi describes the synthesis of abifunctional-antibody-β-galactosidase compound which is cross-linked viaN,N'-o-phenylenedimaleimide. Hamaguchi's compound was reported useful insandwich enzyme immunoassays. Glennie et al., J. Immunol., 139 2367-2375(1987), also describes the linking of two Fab' fragments utilizing thecompound taught in Hamaguchi, i.e., o-phenylenedimaleimide.

Notwithstanding their characterization as bifunctional, when used aspharmaceutical agents, the bifunctional antibodies and bifunctional Fab'compounds of the prior art have the inherent limitation of beingmonofunctional at their site of action. This limitation arises becausethe first of the two specificities of the bifunctional molecule must bedirected to the site of action, i.e., the organ, tissue or antigen ofinterest. This leaves only a single specificity for conferringmono-function to the molecule once it has become immobilized at its siteof action. It is an object of the present invention to develop anintermediate compound, i.e., a multifunctional coupling agent, that issuited for producing a novel series of pharmaceutical agents capable ofbeing trifunctional overall, and thus bifunctional at their site ofaction.

SUMMARY OF THE INVENTION

The present invention is directed to a compound for use as anintermediate in the production of trifunctional antibody-like molecules.The intermediate compound of the present invention functions as atrivalent agent that is capable of coupling two or three Fab'-likefragments. Specifically, the intermediate compound of the presentinvention is a trivalent coupling agent of the formula: ##STR6## whereinX is ##STR7## wherein k×1 or 0; wherein Z is ##STR8## wherein s=1 or 0;wherein n=1 or 0;

wherein q=1 or 0;

wherein Y is ##STR9## wherein Y' is ##STR10## wherein p or m may be thesame or different and are integers ranging from 0 to 20 with theprovisos that when n=0, the sum of m and p is an integer ranging from 1to 20, whereas when n=1, p and m are each an integer that is at least 1and the sum of p and m is an integer ranging from 2 to 20;

wherein R¹ is straight or branched chain lower alkyl having from 1 to 6carbon atoms or lower alkoxy having from 1-6 carbon atoms; and

wherein R² is hydrogen, phenyl, --COOH, or straight or branched chainlower alkyl having from 1-6 carbon atoms, with the proviso that thelower alkyl moiety may be mono-substituted by --NH₂, --OH, or --COOH.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a compound of Formula I aspreviously described. This compound has utility as an intermediate i.e.,a trivalent coupling agent, for use in the formation of bifunctional andtrifunctional antibody-like compounds. By "bifunctional antibody-likecompounds" as used herein is meant compounds having two Fab'-likefragments covalently bonded thereto, preferably having differentspecificities and wherein the Fab'-like fragments substantially retainthe antigen-binding activity of the whole antibodies from which they arederived. By "trifunctional antibody-like compounds" as used herein ismeant compounds having three Fab'-like fragments covalently bondedthereto, preferably having different specificities, wherein theFab'-like fragments substantially retain the antigen-binding activity ofthe whole antibodies from which they are derived. The bifunctional andtrifunctional antibody-like compounds are preferably used as in vivopharmaceutical agents having diagnostic, therapeutic, or a combinationof diagnostic/therapeutic applications.

Structurally, the intermediate compound of the present invention,(hereinafter the "trivalent coupling agent" of the present invention),which is represented by Formula I, has a central moiety "X" from which 3linker arms extend. The central moiety "X" may be a single atom, such ascarbon or nitrogen, or a cyclic molecule that is capable of extendingthree linker arms therefrom. Suitable cyclic molecules may be or containa 5 or 6 membered ring that is aliphatic or may be or contain a 6membered ring that is aromatic. Preferred cyclic compounds are aromatic6 membered rings, such as phenyl. Preferred positioning of the linkerarms on the aromatic ring are at the 1, 3, and 5 positions. However, asthe linker arms become longer, the positioning of the arms on thearomatic ring becomes less critical since steric hinderance at theterminus of the linker arms, due to coupling to the first Fab' likefragment, becomes less of a factor.

The linker arms on the trivalent coupling agent of the present inventionmay be straight or branched chain aliphatic and comprise from about 1-20carbon atoms. In terms of Formula I, the arms are straight chainaliphatic when n=0, s=0, and m and p are each an integer that is atleast 1 and the sum of p and m is an integer ranging from 2 to about 20.Alternatively, the linker arms may be substituted along the way toconfer desirable properties to the linker arms. For example, one or moreof the linker arms may contain one or more amides or ester linkagesalong the chain to confer improved solubility. The amide linkages may beprovided in whole or in part by the various alpha amino acids.

Expressed in terms of Formula I, the presence of a solubility enhancingamide on each linker arm is reflected when in Formula I, n=1 andY=--CONH-- or --NH--CO--. Alternatively, the presence of a solubilityenhancing amide on each linker arm occurs when Z of Formula I is--CONH-- or --NH--CO--. Further, the presence of two solubilityenhancing amide linkages per linker arm occurs when Y of Formula I is--CONH-- or --NHCO-- and when Z of Formula I is independently-CONH-- or--NHCO--.

The use of amino acids, such as serine, lysine, glutamic acid and thelike, which have polar substituents, introduces even greater polarityinto the linker arms of the trivalent coupling agent of the presentinvention, thereby further enhancing its water solubility. The need forpolar substituted amino acids increases as the hydrophobicity of thelinker arms increases, such as with increasing aliphatic chain length.

Since stereochemistry is retained during the synthesis of the compoundsof the present invention (Examples 4-6), specific stereochemistry isintroduced into the linker arms by the use of D or L amino acids.

Analogous to the amide linkages just discussed, each linker arm of thecompound of the present invention may contain from 1-2 ester linkages toenhance the overall solubility of the compound of the present invention.This situation is reflected when Y and/or Z of Formula I areindependently --COO-- or --OCO--. Further it is within the scope of thepresent invention to mix esters and amides in the same linker arms suchas when Z is an amide and Y is an ester of vice versa.

Optionally, one or more of the linker arms may contain a disulfide(--S--S--) linkage. The disulfide linkage may be symmetricallyintroduced by incorporating a cystine residue on each linker arm.However, the use of cystine is not particularly preferred due to thenecessity to utilize blocking reactions to prevent the incorporation ofmultiple residues (i.e., peptide formation). A more preferred method forthe symmetrical incorporation of a disulfide linkage into each linkerarm is to utilize a disulfide containing alpha and omega terminateddiamine of the formula:

    H.sub.2 N--(CH.sub.2).sub.j--h --S--S--(CH.sub.2).sub.h --NH.sub.2

wherein "j" and "h" are independently integers from 2-18 with theproviso that the sum of "j" and "h" is not greater than 20.Alternatively, the structurally analogous alpha and omega terminateddithio is used. The use of these compounds is discussed in detailherein, particularly as they relate to the selective introduction of a--S--S-- linkage in a single linker arm. Expressed in terms of FormulaI, a disulfide linkage occurs when n=1 and Y=--S--S--.

At or near the distal terminus of each linker arm is a maleimide moietyfor binding to a free sulfhydryl group (--SH) on an Fab'-like fragment.The maleimide moiety exhibits selective reactivity with these freesulfhydryl groups at pH 5-8, preferably pH 5-7. However, as the pHincreases towards 8, the maleimide moiety begins to increasingly reactwith free amino groups, such as the ε-amino group of lysine. At pH >8,the selectivity for sulfhydryl begins to decrease. Further, the reactionbetween the maleimide moiety and the amines begin to increase, due toboth the large number of amines and their increasing nucleophilicity.Because of the large number of lysine residues, and thus free aminogroups, on any Fab'-like fragment, the coupling between a maleimidemoiety and any one of the free amino groups at pHs greater than 8 occurswith decreased specificity and may not result in a single reproducibleproduct. In contrast, free sulfhydryl groups on Fab'-like fragmentsoccur at 1-3 specific locations and in the hinge region, unlesschemically modified or genetically engineered to specifically occurelsewhere. Thus, at pH 5-8, preferably 5-7, the reaction between themaleimide moiety and the sulfhydryl groups, which are uniquelypositioned, allow for regiospecific and substantially reproduciblebinding of maleimide moiety at the hinge region or other specificallyadded location. Regiospecific binding at or near the hinge region isimportant because it not only permits the coupling of Fab'-likefragments in a reproducible manner, but is also permits binding to occuraway from the antigen binding portion of the Fab'-like fragment, whichminimizes adverse effects upon the specificity and/or affinity of theantibody.

"Fab'-like fragments" containing free sulfhydryl groups are produced bythe enzymatic cleavage of a whole antibody at its hinge region.Typically enzymatic cleavage of an antibody at the hinge region iseffected either by pepsin or papain. By definition in the art, pepsincleavage of a whole antibody, such as IgG, results in one F(ab')₂fragment and one Fc' fragment. By definition in the art, papain cleavageof a whole antibody under reducing conditions results in two Fabfragments and one Fc fragment. The F(ab')₂ fragment that is obtainedfrom the pepsin cleavage may be reductively cleaved to yield two Fab'fragments. An Fab' fragment is structurally similar to an Fab fragmentin that both fragments contain the intact antigen binding regions of theantibody precursor. However, the Fab' fragment differs from the Fabfragment in that the Fab' fragment is slightly larger having more heavychain. Typically, the Fab' fragment differs further from the Fabfragment by also having one or more additional sulfhydryl groups on itsheavy chain.

Depending upon the species that is the source of the antibody, thenumber of disulfide bridges between the two heavy chains at the hingeregion may vary. As a result, the number of free sulfhydryl (--SH)groups on the Fab and Fab' fragments may also vary from species tospecies. For example, the pepsin cleavage and subsequent reduction ofmouse IgG₁, IgG_(2a) and IgG_(2b) antibody produces mouse Fab' fragmentsthat have three free --SH groups. In contrast, the pepsin cleavage andsubsequent reduction of human IgG₁ antibody produces two Fab' fragmentsthat each have only two free --SH groups. Further, the papain cleavageof the same human IgG₁ results in two Fab fragments, each having only asingle free --SH group. See Nicolotti, et al. U.S. Pat. 4,659,839 atcol. 4 describing this latter cleavage. Human IgG₁ is of interestbecause it is the predominant subclass of monoclonal antibodies used inthe constant region of chimeric antibodies.

The trivalent coupling agent of the present invention couples to Fab'fragments having 1, 2, or 3 free sulfhydryl (--SH) groups. However Fab'fragments having 2 free sulfhydryl groups are especially preferred.

For purposes of the present invention, we collectively define the term"Fab'-like fragments" as including not only those Fab and Fab' fragmentsthat have from 1-3 free sulfhydryl groups on their heavy chain whetherby natural occurrence, chemical modification or genetic engineering butalso as including Fv fragments that have been genetically engineered topossess from 1-3 sulfhydryl groups on either their heavy or light chainor on a combination of both. The "Fv fragment," which is a fragmentderived from either an antibody, an Fab' fragment or an Fab fragment,contains the variable ("v") region of the antibody, which regionprovides specificity for the antigen of interest. In order for agenetically engineered Fv, Fab, or Fab' fragment to be useful in thepresent invention, the sulfhydryl group(s), which are engineered intothe fragment, must be positioned so as not to substantially interferewith antigen binding capacity of the Fv, Fab, or Fab' fragmentrespectively. The determination of the number of the free sulfhydrylgroups in Fab' fragments is well known in the art. Nicolotti, et al.U.S. Pat. No. 4,659,839, which issued on Apr. 21, 1987, describes such amethod using ³ H-(N-ethylmaleimide) and is incorporated herein byreference.

Preferably, the tris-maleimide compound of the present invention is usedto couple Fab'-like fragments that have two free sulfhydryl (--SH)groups. As already disclosed above, such Fab'-like fragments areobtained by the pepsin cleavage and subsequent reduction of human IgG₁.In addition, preferred Fab'-like fragments that have two free sulfhydrylgroups at the hinge region are also obtainable from appropriate human,primate (e.g., chimp) and human-mouse chimeric antibodies. For example,chimeric antibodies having a human constant region yield, upon pepsincleavage and subsequent reduction, Fab'-like fragments that have twosulfhydryl groups in the hinge region. This is because the humanconstant region inherently includes the hinge region as a segment withinit. These chimeric Fab'-like fragments with human constant regions arepreferred for use in humans over Fab'-like fragments from non-humansources because the human constant region substantially reduces thelikelihood of invoking an immune response. This is of paramountimportance when the Fab'-like fragment is intended to be parenterallyadministered to humans as a pharmaceutical agent.

The trivalent coupling agent of the present invention is suited forcoupling two or three Fab' like fragments, to produce an antibody-likecompound for use in diagnostics, therapeutics and/ordiagnostic/therapeutic combinations. By "diagnostics" as used herein ismeant testing that is related to either the in vitro or the in vivodiagnosis of disease states or biological status (e.g., pregnancy,infertility, etc.) in mammals, preferably in humans. By "therapeutics"and "therapeutic/diagnostic combinations" as used herein is respectivelymeant the treatment or the diagnosis and treatment of disease states orbiological status via the in vivo administration to mammals, preferablyhumans, of bifunctional or trifunctional antibody-like molecules thatutilize the trivalent coupling agent of the present invention.

A particularly preferred utility of the coupling agent of the presentinvention is to couple two or three Fab'-like fragments having differentspecificities to produce a bi- or trifunctional antibody-like compoundrespectively. The bifunctional antibody-like compound, which has twodifferent Fab'-like fragments can be used as an in vivo diagnostic ortherapeutic agent. In this utility, the first Fab'-like fragments hasspecificity for the organ, tissue or oncologic antigen of interest,whereas, the second Fab'-like fragment, depending upon utility, hasspecificity either for a diagnostic imaging or dosimetric isotopecomplex (e.g., ¹¹¹ In-ethanolaminethioureabenzyl-EDTA) or for atherapeutic agent (e.g.. a therapeutic radioisotope complex or anantigen coupled to a chemotherapeutic agent).

The trifunctional antibody-like compounds, which are also produced viathe trivalent coupling agent of the present invention have severalutilities. These utilities include use as a diagnostic agent,therapeutic agent or as a combination diagnostic/therapeutic agent. Apreferred utility is as a combination diagnostic/therapeutic agent. Inthis latter utility, the first Fab'-like fragment has specificity for anorgan, tissue or oncologic antigen of interest and binds thereto. Thesecond Fab'-like fragment has specificity for a diagnostic imaging ordosimetric/isotope complex (e.g., a chelated nuclide or paramagneticagent) that permits the imaging of an organ or tissue of interest and/orthe diagnosis of a condition (e.g., cancer) associated with that organor tissue. The third Fab'-like fragment has specificity for atherapeutic agent that can optionally be administered to the patientshould the expected condition present itself to a physician upon theimaging of the organ, tissue or cancer via the immobilized secondFab'-like fragment and its antigen. Thus, the trivalent coupling agentof the present invention enables the production of a trifunctionalpharmaceutical composition that is bifunctional (i.e., both diagnosticand therapeutic) at the site of action.

Alternatively, in its utility as a pure diagnostic agent, the second andthird Fab'-like fragments of the trifunctional antibody-like compoundhave specificity for the same or different diagnostic imaging ordosimetric complexes. Whereas, in its utility as a pure therapeuticagent, both the second and third Fab'-like fragments of thetrifunctional antibody-like compound have specificity for the same ordifferent therapeutic agents.

There are instances when a trifunctional antibody like compound ("TFA")having specificities for two different tumor antigens might be useful.There is some evidence that melanoma may express either the p96.5 or thegp240 antigen or both. If this is true, then a TFA with bindingspecificities directed toward these two antigens in conjunction with ananti-chemotherapeutic, anti/imaging, or antitherapeutic isotope might beuseful. A similar situation may exist in lung cancer, with some tumorsexpressing the KS1/4 antigen or CEA, or both. Thus, in its utility aseither a diagnostic or as a therapeutic agent, the first and secondFab'-like fragments have specificities for the different antigensexpressed on or by the same tumor or tissue.

An Fab'-like fragment can have specificity for a therapeutic agenteither directly or indirectly. Specificity for a therapeutic agent is"direct" when the Fab' fragment is specific for the therapeutic agentitself. Specificity for a therapeutic agent is "indirect" when the Fab'fragment is specific for a select antigen or hapten to which thetherapeutic agent is coupled. When an Fab'-like fragment has specificityfor a select antigen or hapten one can vary the number and type oftherapeutic agent attached to the select antigen or hapten, therebypermitting the treating physician to vary the treatment depending uponfactors such as the condition presented, the severity of the condition,the patient's sensitivity to particular pharmaceuticals, and thepresented condition's response to certain pharmaceuticals. One couldeven co-administer two therapeutic agents bound to the same selectantigen or hapten to provide a localized synergistic effect at theorgan, tissue, or tumor of interest.

Examples of therapeutic agents capable of "directly" binding to anFab'-like fragment include chelate complexes that are formed betweenchelating agents and chelatable radionuclides that are β⁻ emitters.Suitable chelating agents for the radionuclides (and/or the paramagneticmetal ions used in diagnosis) are polyacidic organic molecules thatfurther contain organic nitrogen, phosphorous, oxygen or sulfur. By wayof example, suitable chelating agents include ethylenediaminetetraaceticacid ("EDTA"); ethanolaminethioureabenzyl-EDTA ("EOTUBE");diethylenetriaminepentaacetic acid ("DTPA"); methylthioureabenzyl DTPA("MeTUBD"); 1,4,7,10-tetrazacyclododecane-N',N",N'",N""-tetraacetic acid("DOTA"); L-aminobenzyl-EDTA, and the like. Other suitable organicchelating agents are disclosed in Gries et al. U.S. Pat. 4,647,447 whichis incorporated herein by reference. Suitable β⁻ emitters are thechelatable ions of ⁶⁷ Cu, ¹⁸⁶ Rh, ¹⁸⁸ Rh, ¹⁸⁹ Rh, ¹⁵³ Sm, ⁹⁰ Y, and ¹¹¹In (Auger).

Examples of therapeutic agents capable of becoming "indirectly" bound byan Fab'-like fragment are compounds of the formula:

    Substrate-Cytotoxic Agent

wherein the "substrate" has at least one epitope bindable by theFab'-like fragment and is the substrate for an enzyme or an activefragment thereof, and wherein reaction of the enzyme with the boundSubstrate-Cytotoxic Agent cause release of the cytotoxic agent from thesubstrate.

The term "Cytotoxic Agent" as used herein means compounds that areuseful in the treatment of neoplasms, whether benign or malignant. Suchdrugs include, in general, alkylating agents, antiproliferative agents,tubulin-binding agents, cytotoxins in general, and the like. Preferredclasses of such compounds are the nitrogen mustard agents, the vincaalkaloids, the daunomycin family, the mitomycins, the bleomycins, thecytotoxic nucleosides, the pteridine family of drugs, thepodophyophyllotoxins, the sulfonylureas (as described in European PatentPublication No. 222,475, published May 20, 1987), and lowmolecular-weight toxins such as the trichlothecenes and the colchicines.Particularly preferred members of those classes include, for example,doxorubicin, daunorubicin, aminopterin, methotrexate methopterin,dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil,6-mercaptopurine, cytosine arabinoside, podophyllotoxin, etoposide,melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine,trichothecene, desacetylcolchicine, and the like.

The conversion of trivalent antibody-like compounds into diagnostic,therapeutic and combination diagnostic/therapeutic agents is discussedin more detail in our copending U.S. patent application Ser. No.07/491,406, cofiled herewith.

As already pointed out, the trivalent coupling agent of the presentinvention is suited for coupling to Fab'-like fragments having one, two,or three, free sulfhydryl groups (--SH) i.e., sulfhydryl moieties.However, the number of trivalent coupling agents of the presentinvention that are required to form a trifunctional antibody-likecompound will vary from 1-2 depending upon the number of free sulfhydrylgroups on the Fab' fragments. For example, three Fab' fragments eachhaving a single free --SH group, require only one trivalent couplingagent of the present invention to accomplish coupling to produce atrifunctional antibody-like molecule. In contrast, three Fab'-likefragments (designated as F₁ ab, F₂ ab, F₃ ab'), each having two freesulfhydryls, would usually require two trivalent coupling agents of thepresent invention to give rise to a trifunctional antibody-likecompound. Likewise, three Fab'-like fragments each having three freesulfhydryl groups would require two trivalent coupling agents of thepresent invention. Moreover, two or three Fab'-like fragments withdifferent numbers of free sulfhydryls could also be combined to producea bifunctional or trifunctional antibody-like molecule respectively.However, it is most preferred to use trivalent coupling agents of thepresent invention to couple two or three Fab'-like fragment wherein eachfragment has two free sulfhydryl groups.

Scheme I generically provides the reaction sequences for using thetrivalent coupling agent, XI, of the present invention, wherein XI is aschematic representation of the compound of Formula I, to couple threeFab'-like fragments each having two free sulfhydryl groups. Thetrivalent coupling agent, XI, may be dissolved in a small volume (<10%of the reaction volume) of organic solvent to which is added theFab'-like fragment to be derivatized. In Scheme I, a molar excess,preferably at least 10 fold, more preferably at least 20 fold, mostpreferably at least 30 fold, of XI is combined in an aqueous buffer atpH 5-8, preferably pH 5-7, with a first Fab'-like fragment, F₁ ab'. Inthe ensuing reaction, two of the three terminal maleimides on thetrivalent coupling agent, XI, react with the two free sulfhydryl groups(--SH) of the first Fab'-like fragment, F₁ ab', to produce a coupledproduct XII having only a single reactive maleimide moiety remaining.The proximity of the two sulfhydryl groups (--SH) on the Fab'-likefragment and the relative position of the linker arms on the couplingagent permits the second maleimide moiety of the coupling agent toinstantaneously couple to the second sulfhydryl (--SH) group once thefirst coupling has taken place. The coupled product XII is thenseparated by gel filtration from excess XI whereupon XII is reacted inan aqueous buffer at pH 5-8, preferably pH 5-7, with a second Fab'-likefragment designated F₂ ab' and having a second specificity. Theresultant product XIV is a bifunctional Fab'-like moiety having a singlefree sulfhydryl group (--SH) for coupling with a second trivalentcoupling agent of the present invention. In a separate reaction shown inScheme I, a third Fab'-like fragment, F₃ ab', having a thirdspecificity, is reacted as described above in aqueous solution at pH 5-8, preferably pH 5-7, with a molar excess of the trivalent coupling agentof the present invention, XV, which may be the same or different thanXI. For example, unlike XI, XV may have labile linker arms that containa disulfide linkage (--S--S--) within each of the arms. Alternativelythe linker arms of XV may vary from the linker arms of XI in length,polarity, or a variety of other factors to accommodate the Fab'-likefragment being coupled. As previously described for XI, the trivalentcoupling agent, XV, may be dissolved in a small volume (<10% of theaqueous reaction volume) of organic solvent to which is added theFab'-like fragment to be derivatized. In Scheme I, the coupling of F₃ab' to XV produces a compound XVI having a single free maleimide moiety.##STR11## The subsequent reaction of product XIV and XVI in aqueoussolution at pH 5-8 produces a trifunctional antibody-like compound XVIIthat incorporates the trifunctional coupling agent of the presentinvention. The generic use of the trivalent coupling agent of thepresent invention to prepare trifunctional antibody-like compounds ismore fully discussed in Example 12 herein. Specific uses are describedin Examples 13 and 14.

The trivalent coupling agent of the present invention can be synthesizedfrom a variety of compounds acting as the central moiety. Preferably thecompound acting as the central moiety "X" has three chemical functionalgroups that are capable of binding a maleimide moiety either directly orindirectly Suitable chemical functional groups include alcohols,aldehydes, carboxylic acids, esters, amides, amines and the like. Thecentral moiety "X" may be directly bound to the three chemicalfunctional groups (e.g., 1,3,5-benzene tricarboxylic acid).Alternatively, the central moiety may contain one or more carbon,nitrogen, oxygen, sulfur or phosphorus atoms, that act as linkersbetween the central moiety and the three chemical functional groups. Forexample, in tris(2-aminoethyl)amine, the three C₂ (ethyl) chains act asarms between the central moiety, an amino nitrogen, and the three-NH₂groups at their terminus. In its simplest mode, the trifunctionalcoupling agent of the present invention is synthesized from a triaminocompound, such as tris(2-aminoethyl)amine, andN-methoxycarbonylmaleimide.

The trivalent coupling agent of the present invention can have a varietyof arm lengths that depend upon both the steric requirements of theFab'-like fragments being linked and the conformational requirements ofthe trifunctional antibody-like compound ultimately being synthesized.In general, slightly longer linker arms are preferred in the synthesisof trifunctional antibody-like compounds than in the synthesis ofbifunctional antibody-like compounds. The longer linker arms apparentlyreduce steric hinderance in the hinge region allowing access tounderivatized sulfhydryls during additions of the Fab'-like fragmentwith its third specificity.

Typical compounds used to increase the length of the linker arms are thealpha and omega terminated diamines and/or dithiols. Of these, the alphaand omega terminated dithiols are preferred. By "alpha and omegaterminated diamines" as used herein is meant diamines of the formula:

    H.sub.2 N--(CH.sub.2).sub.x --NH.sub.2

wherein the subscript "x" is an integer ranging from 2 to about 20. By"alpha and omega terminated dithiols" as used herein is meant dithiolsof the formula:

    HS--(CH.sub.2).sub.t --SH

wherein the subscript "t" is an integer ranging from 2 to about 20.

The alpha and omega terminated dithiols are preferably used to increasethe length of a linker arm of the trivalent coupling agent of thepresent invention after it has already been coupled to its firstFab'-like fragment. This is particularly important where the maleimideis bound to an expensive Fab' fragment and it is determined that asubsequent reaction of the maleimide with a second Fab' fragment issterically hindered. This utility and the method of combining isreflected in Scheme II. For example in Scheme II, an excess amount ofthe trivalent coupling agent of the present invention XX is reacted atpH 5-8, preferably pH 5-7, with a Fab'-like fragment XXI, having twofree sulfhydryl groups, to form a compound XXII having a singlemaleimide moiety available for further coupling. To further increase thelength of the uncoupled linker arm on XXII, an excess amount of an alphaand omega terminated dithiol XXIII, as defined herein, is reacted withthe maleimide moiety of XXII to form the omega terminated thiol XXIV.Reaction at pH 5-8, preferably pH 5-7 of the thiol XXIV with an excessof a bis-maleimide type compound XXV, such as p-phenylenedimaleimide,produces a coupling agent complex XXVI having an extended linker armwith a reactive maleimide moiety at its terminus. The complex of XXVI isanalogous to the complex XXII. However, the former's longer linker armserves to minimize or eliminate any stearic hinderance associated withthe coupling agent's binding of the first Fab-like fragment.

As a practical matter, a trivalent coupling agent of the presentinvention that has short linking arms selectively couples to a singleFab'-like fragment having two free --SHs in high yield. However,subsequent coupling is sterically hindered, and thus prevented, untilthe length of the uncoupled arm is increased so as to reduce oreliminate the steric hinderance. Once the length of the linker arm isincreased such as described in Scheme II, the second Fab'-like fragmentcan be selectively coupled.

Thus, the present invention also encompasses a method for extending thelength of one of the linker arms of the compound of Formula I, whichmethod comprises: ##STR12##

(a) combining the compound of Formula I at pH 5-8 with an Fab'-likefragment having at least 2 free sulfhydryl groups, whereby amaleimide-Fab' complex having a single reactive maleimide moiety isformed;

(b) combining the maleimide-Fab' complex from Step (a) at pH 5-8 with acompound of the formula:

    HS--(CH).sub.2).sub.t--SH                                  II

wherein the subscript "t" is an integer from 2 to about 20, the compoundof Formula II being combined in an amount effective to preventintermolecular crosslinking between the Fab'-maleimide complexes, thecombination forming a coupled product between the maleimide-Fab' complexand the compound of Formula II, the coupled product having an extendedlinker arm with an --SH moiety extending distally therefrom; wherebysaid single reactive maleimide moiety becomes coupled to one of the --SHmoieties on the compound of Formula II, forming a maleimide-Fab' complexwith a single extended linker arm. Although the couplings in Steps (a)and (b) above may be performed at pH 5-8, they are preferably performedat pH 5-7.

In Step (b), the amount of the compound of Formula II that is effectiveto prevent intermolecular crosslinking between molecules of themaleimide-Fab' complex is dependent upon a number of factors includingthe size of the compound of Formula I, and the size of the integer "t"in Formula II. Typically, increasing molar ratios of the compound ofFormula II relative to the maleimide-Fab' complex result in decreasingintermolecular crosslinking of the complex by the compound of FormulaII. One skilled in the art can determine the minimum mole ratio of thecompound of Formula II relative to the maleimide-Fab' complex to preventcrosslinking of the maleimide-Fab' complex. Preferably, howwever, a 10fold or greater molar excess of the compound of Formula II is employedin Step (b) to prevent crosslinking.

The present invention also encompasses the above method furthercomprising the step of:

(c) combining the coupled product of Step (b) at pH5-8, preferably 5-7,with an amount of a bis-maleimide effective to prevent intermolecularcrosslinking between molecules of said coupled product, whereby amaleimide-Fab' complex that has an extended linker arm with a reactivemaleimide at its terminus is formed.

By "bis-maleimide" as used in Step (c) above is meant a compound havinga maleimide moiety at each of two termini. Typical bis-maleimidesinclude N,N'-o-phenylenedimaleimide, N,N'-p-phenylenedimaleimide, andN,N'-(oxydimethylene)dimaleimide, which compounds are well known in theart. See, for example, Weston et al., Biochemica et Biophysica Acta.612: 40-49 (1980). Preferred bis-maleimides includeN,N'-bis(maleimidopropionyl)-2-hydroxy-1,3-propanediamine ("BMP") andbis-(maleimido)-methyl ether ("BMME"). BMP is commercially availablefrom Sigma Chemical Co., St. Louis, Mo., and BMME is commerciallyavailable from Boehringer Mannheim Corp., Indianapolis, Ind.

In Step (c), the amount of bis-maleimide that is effective to preventintermolecular crosslinking between molecules of the "coupled product"is dependent upon a variety of factors. Generally, increasing molarratios of the bis-maleimide relative to the coupled product of Step (b)result in decreasing intermolecular crosslinking of the coupled productby the bis-maleimide. To determine the minimal amount of bis-maleimidenecessary to prevent crosslinking, one skilled in the art can run aseries of reactions with increasing molar ratios of bis-maleimiderelative to coupled product until significant crosslinking is no longerobserved. Preferably to avoid crosslinking, one simply employs a 10 foldor greater molar excess of bis-maleimide relative to coupled product.

As previously discussed herein, Fab'-like fragments having at least twofree sulfhydryls are derived from mouse IgG₁, IgG_(2a) ; and IgG_(2b) ;human IgG; and primate (e.g., chimp) IgG.

The lability of all three of the linker arms on the trivalent couplingagent of the present invention are increased when a labile chemicalfunctional group,. such as a disulfide (--S--S--) is symmetricallyintroduced into each arm. Chemical reactions symmetrically introducing a--S--S-- moiety into each linker arm are well known to those skilled inthe art. In order to selectively increase the lability of a singlelinker arm, a labile bond, preferably a --S--S--, must be selectivelyincorporated into that single linker arm. Selective incorporation isbest achieved after an initial coupling between a trivalent couplingagent of the present invention and an Fab'-like fragment having two freesulfhydryls. As a result of this coupling, there remains a singleterminal maleimide moiety for further reaction. The labile disulfidelinkage (--S--S--) is then incorporated into the uncoupled linker armvia an alpha-omega diamine or more preferably an alpha-omega dithiollike compound that contains a disulfide moiety therein. The alpha-omegadithios having a disulfide therein are preferred over the correspondingdiamines due to the minimization of undesirable side reactions with thefree amines on the Fab' like fragments at the reaction pH.

By way of example, disulfide containing dithiols and diamines arecompounds of the respective formulas:

    HS--(CH.sub.2).sub.g-f ----S--S--(CH.sub.2).sub.f --SH; and

    H.sub.2 N--(CH.sub.2).sub.j-h --S--S--(CH.sub.2).sub.h -NH.sub.2

wherein g and j are integers from 2-18 and f and h are integers from2-18 with the proviso that the sum of g and f and/or j and h is notgreater than 20. Selective incorporation of a disulfide containingdithiol into a single linker arm at pH 5-8, preferably pH 5-7, ispreferably accomplished via the reaction sequences already provided inScheme II.

The following examples are given by way of illustration only and shouldnot be construed as limiting the invention in spirit and/or scope.

DESCRIPTION OF THE PREFERRED EMBODIMENT Example 1 Preparation ofN-Methoxycarbonylmaleimide ##STR13##

To a 1000 ml 3-neck round bottom flask was added 400 ml of ethylacetate. The flask was placed in an ice bath and the temperature wasallowed to drop to about 0° C. To the cooled flask was sequentiallyadded with stirring 7.76 g of maleimide and 8.8 ml ofN-methylmorpholine. Then, through an addition funnel was added to thestirring mixture 6.26 ml of methyl chloroformate at a rate so as not toraise the temperature above 3° C. Through the addition funnel was thenadded 5 ml of ethyl acetate as washing and the washing was added to thereaction mixture. The reaction mixture was stirred for 30 minutes atbetween 0°-3° C. Thereafter, the reaction mixture was filtered through aBuchner funnel. The flask was washed 2× with 10 ml of ethyl acetate andthe washings were also filtered. The resulting precipitate was washed2×with 10 ml of ethyl acetate. The combined filtrate and washings wereextracted with 100 ml of ice cold water, dried (10 g Na₂ SO₄), andevaporated to dryness under reduced pressure. The residue wasredissolved in 50-100 ml of ethyl acetate:isopropylether (40:60/v:v)using a water bath at 60° C. The resulting solution was filtered, cooleduntil crystals appeared. After 30 minutes in an ice bath, the cooledsolution and crystals were filtered through a sintered glass funnel andthe crystals washed 2× with 20 ml of isopropyl ether. The crystals werevacuum dried overnight. M.P. 61°-63° C.

Example 2 Tris-(2-N-maleimidoethyl)amine ("TMA") ##STR14##

To 15 g of NaHCO₃ in a 250 ml Erlenmeyer flask was added 100 ml of coldwater and the mixture was stirred in an ice bath until the reactionmixture was at 0° C. To 80 ml of the supernatant solution in a 100 mlround bottom flask was added 1.8 ml of tris(2-aminoethyl)amine and themixture was cooled 0° C. in an ice bath. To the cooled reaction mixturewas added with stirring 6.2 g of finely groundN-methoxycarbonylmaleimide and the mixture was stirred for an additional10 minutes in the ice bath. Thereafter, 240ml of water was added to themixture and it was stirred at room temperature for 30 minutes. Then, thepH of the solution was adjusted to between pH 6.7 with concentrated HCland the volume was reduced to 100 ml by evaporation under reducedpressure. The pH of the resulting solution was adjusted to 10 withsaturated Na₂ CO₃ solution. The resultant solution was extracted 3× with200 ml of ethyl acetate and the combined organic phases were washed 2×with 100 ml of H₂ O. The organic phase was dried (30 g Na₂ SO₄),filtered and evaporated under reduced pressure to dryness. The residuewas dissolved in 40 ml of warm ethyl acetate, filtered (Buchner funnel),and evaporated under reduced pressure to dryness. The residue wasdissolved at a ratio of 5 ml/g (residue) in ethyl acetate:methylenechloride (1:3/v:v). To a 150 ml Lobar silica gel column that had beenpre-equilibrated with 2 bed volumes of ethyl acetate:methylene chloride(1:3/v:v) was added a 5 ml aliquot of the dissolved residue. The columnwas eluted with the same solvent at 4 ml/min and the TMA fraction, asmonitored at A₂₈₀, was collected in a 500 ml round bottom flask. The TMAfraction was evaporated to dryness. Additional 5 ml aliquots of thedissolved residue were similarly treated and the corresponding TMAfractions were evaporated by dryness. The TMA residues were dissolved in10 ml of the elution solvent, pooled together and evaporated underreduced pressure to dryness. The combined residue was dissolved in 40 mlof ethyl acetate:isopropyl ether (3:1/v:v) using a 60° C. water bath,filtered, and cooled sufficiently until the TMA precipitated out ascrystalline yellow needles. The resultant TMA crystals were collected ona sintered glass funnel, washed 2× with 5 ml of isopropyl ether, anddried overnight under vacuum, M.P. 132°-133° C.

Analysis for C₁₈ H₁₈ N₄ O₆ (MW=386.36).

Calcd: C, 55.95; H, 4.70; N, 14.50.

Found: C, 55.54; H, 4.69; N, 14.45.

¹ H NMR δ_(TMS) CDCl₃ (300 MHz): 6.65(6H,s); 3.49(6H,t); and 2.68(6H,t).

I.R. (KBr): 1700 cm⁻¹ (C═O).

U.V. (DMF): peak at 272, molar extinction coefficient=1920.

Example 3 ##STR15##

One mole of cyclohexan-1,3,5-triol is reacted with 3 moles of BrCH₂ COO⁻K⁺ in tetrahydrofuran in the presence of 3 eq. of potassium t-butoxideat 22° C. for 1 hr. The resulting tricarboxylic acid is then reactedwith N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) for 1hr at 0° C. in tetrahyrafuran to yield the tris-succinimide. Thiscompound will then react with a 10 fold excess of ethylenediamine insaturated sodium bicarbonate for 1 hr. at 22° C. The resultant compoundis then reacted with N-methoxycarbonylmaleimide (whose synthesis isdescribed in Example 1) in saturated sodium bicarbonate at 0° C. for 10minutes yielding a tris-maleimide in which the X component of Compound Iis a cyclohexyl moiety.

Example 4 ##STR16##

To 1 mole of serine in aqueous solution in the presence of NaHCO₃ at 0°C. is added 1.1 moles N-methyoxycarbonylmaleimide. The reaction isallowed to proceed at 0° C. for 10 minutes followed by a 30 minuteincubation at room temperature. The resulting product,2-(N-maleimido)-3-hydroxypropanoic acid, is then reacted in diglyme witha 10% molar excess of N-hydroxysuccinimide in the presence ofdicyclohexylcarbodiimide at 0° C. for 1 hr. followed by incubation for 3hrs. at room temperature to yield the succinimide esters of thepreviously mentioned acid. The succinimide ester compound is thenreacted with tris(2-aminoethyl)amine in dimethylformamide (DMF) at roomtemperature for 4 hr. to produce the tris-maleimido compound, having thestructure shown above. Throughout the above described reactions, theinitial stereo chemistry of the amino acid starting material ismaintained about its asymmetric center (*). Hence, by selection ofL-serine or D-serine, L or D stereochemistry respectively is maintainedabout each asymmetric center.

Example 5 ##STR17##

To an aqueous bicarbonate solution which has been cooled to 0° C. isadded lysine in which the terminal amino group is protected by at-butoxycarbonyl (BOC) group. To this is then added approximatelyequimolar amounts of N-methoxycarbonylmaleimide. The reaction is allowedto proceed for 10 min. at 0° C. followed by a second incubation at roomtemperature for 30 min. The resultant α-maleimido carboxylic acid isthen be reacted in tetrahydrofuran with a 10% molar excess ofN-hydroxysuccinimide in the presence of N,N-dicyclohexylcarbodiimide at0° C. for 1 hr. followed by an incubation at room temperature for 3 hrs.to yield the succinimide ester of the aforementioned acid. Thesuccinimide ester is then reacted with tris(2-aminoethyl)amine indimethylformamide (DMF) for 4 hrs. at room temperature to yield atris-maleimide compound wherein the terminal amine still bears the BOCprotecting group. Removal of the BOC group is accomplished by hydrolysisfor 1 hr with 3M HCl in ethyl acetate at room temperature. As in Example4, stereochemistry about the asymmetric center (*) in the lysinestarting material is retained in the final product.

Example 6 ##STR18##

To an alpha amino acid such as lysine, in which the terminal amino groupis free and the alpha amino group is protected by a phenoxycarbonyl(POC) group, is condensed a trialdehyde compound of the formula R(CHO)₃in the presence of NaBH₃ CN. The POC protecting groups on the resultanttricondensation product are removed by hydrogenation over Pd/C. Theresulting deprotected alpha amino groups of the tricondensation productare now suited for reaction with N-methoxycarbonyl maleimide. Thetricondensation product is dissolved in aqueous bicarbonate at about 0°C. To this is added N-methoxycarbonylmaleimide and the temperature ismaintained at 0° C. for 10 min. Thereafter, the reaction is allowed toproceed at room temperature for 30 min. The resultant maleimide is thenpurified as described for TMA (Example 2 herein). As for Examples 5 and6, any stereochemistry about the asymmetric center (*) in the lysinestarting material is retained in the final product.

Example 7 ##STR19##

To 1,3,5-benzene tricarboxylic acid in tetrahydrofuran (THF) at 0° C. isadded a 10% equivalent excess of N-hydroxysuccinimide in the presence ofdicyclohexylcarbodiimide. The reaction mixture is maintained at 0° C.for 1 hour followed by an incubation for 3 hr. at room temperature toyield the tris-succinimidyl ester of the triacid.

In a separate reaction ethylene diamine is reacted according to theprocedure in Example 2 with an equimolar amount or less ofN-methoxycarbonylmaleimide, which is prepared according to the procedurein Example 1. The resulting product, 2-N-maleimidoethylenediamine, isthen reacted with the tris-succinimidyl ester from above in a saturatedsodium bicarbonate solution for about 1 hr. at 22° C. to produce thetris-maleimide corresponding to the chemical formula shown above.

Example 8 Tris[2-N-(maleoylglycyl)aminoethyl]amine ("TMG") ##STR20##

Glycine (1.5 g, 20 mmol) in saturated NaHCO₃ (100 ml) was vigorouslystirred at 0° C. with finely ground N-methoxycarbonylmaleimide (3.1 g,20 mmol). After 10 minutes the solution was diluted with 400 ml of waterand stirred at room temperature for 40 min. The pH of the solution wasadjusted to ˜7 with concentrated HCl and the neutralized solution wasevaporated in vacuo to about 50 ml. Thereafter, the solution wasacidified to pH ˜2 with 3N HCl and extracted two times with 100 ml ofethyl acetate. The combined ethyl acetate extract was washed with water,dried over anhydrous Na₂ SO₄ and evaporated to dryness. The crudeproduct was dissolved in 5 ml of CH₂ Cl₂ :CH₃ COOH/95:5 and passedthrough a silica gel flash column (60 g) eluted with the same solvent.After evaporation of the organic solvent the product was lyophilized toyield 1.85 g of fluffy white maleoylglycine.

¹ H NMR (D₂ O): δ4.30(2H, s), and 6.95(2H, s).

IR (KBr) 1710 cm⁻¹ (C═O)

Maleoylglycine (1.55 g. 10 mmol) in 50 ml of diglyme was treated at 0°C. with N-hydroxysuccinimide (1.27 g, 11 mmol) anddicyclohexylcarbodiimide (2.23 g. 11 mmol). After 1 hour at 0° C. and 3hours at room temperature, the reaction mixture was filtered andevaporated to dryness to yield 1.9 g of crude maleoylglycineN-succinimidyl ester.

Maleoylglycine N-succinimidyl ester (1.9 g 7.5 mmol) was dissolved in 35ml of DMF, and tris(2-aminoethyl)amine (329 mg, 2.25 mmol) in 10 ml ofdimethylformamide (DMF) was added dropwise with stirring to thesolution. The reaction was monitored by reverse phase (C₁₈) highpressure liquid chromatography (HPLC) eluted with a linear gradient from80% 0.1M ammonium acetate (pH 5)/20% CH₃ OH to 50% 0.1M ammonium acetate(pH 5)/50% CH₃ OH. The reaction solution was concentrated and the crudeproduct ("TMG") was divided into three portions and purified on a 150 mlLobar reverse phase C₁₈ column eluted with a step gradient from 20% CH₃OH/80% 0.1M ammonium acetate, pH 5 to 50% CHOH/50%s 0.1M ammoniumacetate.

¹ H NMR (DMF): δ2.50(6H); 3.18(6H); and 7.05(6H).

Example 9 Tris[2-N-(maleoylglycylglycyl)aminoethyl]amine ("TMGG")##STR21##

To 660 mg of glycylglycine (5 mmol) in 25 ml of saturated NaHCO₃ at 0°C. was added 775 mg of N-methoxycarbonylmaleimide (5 mmol) with vigorousstirring. After 10 minutes, the solution was diluted with 100 ml ofwater and stirred at room temperature for 30 minutes. The pH of thesolution was adjusted from 8 to 6.3 with 3N HCl and the solution wasevaporated with rotation under reduced pressure ("rotavaped") toapproximately 20 ml. The concentrated solution was acidified to pH 2with 3N HCl and extracted two times with 50 ml of ethyl acetate. Thecombined ethyl acetate extract was dried over anhydrous Na₂ SO₄ andevaporated to dryness in vacuo.

The resulting residue was dissolved in 18 ml of diglyme and treated at0° C. with N-hydroxysuccinimide (460 mg 4 mmol) anddicyclohexylcarbodiimide (824 mg, 4 mmol). After stirring 1 hour at 0°C. and 2 hours at room temperature, the reaction mixture was filteredand the filtrate was evaporated to dryness to yield 3.2 mmolmaleoylglycylglycine N-succinimidyl ester.

To 66 mg of tris(2-aminoethyl)amine (0.45 mmol) in 2 ml ofdimethylformamide ("DMF") was added dropwise with stirring 8 ml of 0.2Mmaleoylglycylglycine N-succinimidyl ester in DMF. After stirring at roomtemperature for 1 hr., the solution was rotavaped to 3 ml and passedthrough a silica gel flash column (60 g) eluted sequentially with (1)150 ml EtOAc:MeOH/90:10; (2) 150 ml EtOAc:MeOH/75:25; (3) 150 mlEtOAc:meOH/50:50; and (4) 150 ml MeOH. After evaporation of the solvent,0.22 g of TMGG as a light yellow residue was obtained from fraction 3.

¹ H NMR (DMF): δ2.55 (6H); 3.25(6H); 3.95(6H); 4.35(6H); and 7.10(6H).

Example 10 Preparation Of Antibodies (a) Anti-In-EDTA ("CHA")

The antibody herein designated as "CHA" is a monoclonal anti-haptenantibody having specificity for the complex formed betweenethylenediaminetetraacetic acid ("EDTA") and the indium (III) ion. Forimaging purposes, the ¹¹¹ In isotope of indium (III) is used. In thepresent invention, the EDTA derivative, ethanolaminethioureabenzyl EDTA("EOTUBE") was used as the chelating agent. The CHA 255 antibody wasprepared as follows. Spleen cells from BALB/c mice multiply immunizedwith the antigen were fused with a variant of the P3.653 myeloma cellline. See Gerhard, Monoclonal Antibodies, edited by Kenneth et. al.,Plenum Press, New York (1980). The resulting hybridomas were screened bya solid phase second antibody radioimmunoassay for their ability tobinding indium aminobenzyl-EDTA (Wang et. al., Journal of ImmunologicalMethods, 18, 157 (1977)). Based on their high titers and relatively highaffinity as determined by inhibition of binding by unlabeled antigen, amonoclonal antibody designated as CHA 255 was chosen for further studyand injected intraperitoneally into BALB/c mice for ascites production.The monoclonal antibodies were purified from mouse ascites byion-exchange chromatography on DEAE-cellulose as described by Parham etal., J. Immunol. Meth., 53, 133 (1982). Monoclonal antibody CHA 255 isfurther described by Reardon, D. T., et. al., Nature, 316, p. 265-268(1985) and Meares et. al., U.S. Pat. No. 4,722,892, issued Feb. 2, 1988,herein incorporated by reference. Hereinafter, the CHA 255 antibody isreferred to as "CHA."

(b) Anti-Y-DTPA ("CYA")

The antibody designated herein as "CYA" is a monoclonal anti-haptenantibody having specificity for the complex formed between the chelatingagent, diethylenetriaminepentaacetic acid ("DTPA"), and the yttrium(III) ion. For therapeutic purposes the ⁹⁰ Y isotope of yttrium (III) isused. For enhanced pharmaceutical acceptability, themethylthioureabenzyl derivative of DTPA, which is known asmethylthioureabenzyldiethylenetriaminepentaacetic acid ("MeTUBD") wasused. The CYA316 antibody (hereinafter "CYA") was prepared using thegeneral techniques described in Reardon. et al., "Antibodies AgainstMetal Chelates," Nature, 316: 265-267 (1985) and in Meares. et al. U.S.Pat. No. 4,722,892, the latter being incorporated herein by reference.

(c) Anti-CEA ("ZCE")

The antibody designated herein as "ZCE" is a monoclonal antibody havingspecificity for carcinoembryonic antigen. The "ZCE" antibody iscommercially available from Jean Pierre Mach, University of Lausanne,Lausanne, Switzerland.

(d) Chimeric Anti CEA ("xCEM")

The antibody designated herein as "xCEM" is a mouse/human chimericantibody having specificity for carcinoembryonic antigen. The "xCEM"antibody was cloned and expressed according to the procedure taught inBiedler et al., J. Immunol., 141 pp. 4053-4060 (1988).

(e) Chimaric Anti-In-EDTA ("xCHA")

The antibody designated herein as "xCHA" is a mouse human chimericantibody having specificity for the In-EDTA chelate complex. The "xCHA"antibody was prepared by essentially the same method used for thepreparation of "xCEM" above (i.e., J. Immunol., 141: pp. 4053-4060(1988)) except that in the preparation of "xCHA," the variable regionsfrom the murine antibody CHA-255 were used instead of the variableregions from the murine antibody CEM-231. The synthesis of xCHA isfurther described in U.S. patent application Ser. No. 07/274,106, by M.J. Johnson, filed 11/17/88, herein incorporated by reference, and wasdescribed in a presentation to the 7th International Congress ofImmunology, Berlin, Aug. 1, 1989.

Example 11 Preparation Of The Bifunctional Antibody-Like compoundxCEM-TMA-xCEM (a) Preparation Of xCEM-Fab'SH

A chimeric monoclonal antibody to carcinoembryonic antigen (CEA) anddesignated as "xGEM" was digested with pepsin using the conventionaltechnique described in Example 12 (a) herein, except that the digestionwas for only 3 hours. (See, also for example, Nicoletti et al, U.S. Pat.4,659,839, which details pepsin digestion and is incorporated herein byreference.) The digested xCEM was then dialyzed overnight against boratebuffer, pH ˜8 (by paper). The absorbance of the dialyzed pepsindigestate was measured at 280 nm ("A₂₈₀ ") which indicated 3.9 mg/ml ofprotein. To 900 μl of the dialyzed xCEM digestate was added 2 μl of 0.5Mdiethylenetriaminepentaacetic acid (DTPA) and the mixture was incubatedat 37° C. for 15 minutes. Thereafter, 36 μl of 0.5M cysteine (Cys) wasadded to the mixture which was incubated for a further 10 minutes at 37°C. The resultant reaction mixture was applied to a Biogel P-6 column(Biorad Laboratories, Richmond, Calif. 94804) and 2.0 ml of a proteinfraction were collected. The fraction's A₂₈₀ indicated a 1.3 mg/ml or 26μM concentration of the Fab'-like fragment designated as xCEM-Fab'SH.The concentration of sulfhydryl groups in the fraction was determinedusing the standard technique of adding a molar excess of5,5'-dithiobis-(2-nitrobenzoic acid) (i.e., DTNB) plus borate bufferedsaline (50 mM sodium borate, 50 mM NaCl, pH 8.2) to an aliquot of thefraction and measuring the absorbance difference at 412 nm between theDTNB containing aliquot and a blank. Using this technique, the number ofsulfhydryl groups per Fab' fragment was calculated to be 1.5, indicatingapproximately 1.5 sulfhydryl groups per xCEM-Fab'SH.

(b) coupling Tris-(2-N-malaimidoethyl)amine With xcEM-Fab'SH

To 1.0 mg of tris-(2-N-maleimidoethyl)amine (i.e., "TMA") dissolved in80 μl of dimethylformamide was added 1 ml of the eluate containingxCEM-Fab'SH from above. The reaction mixture was allowed to stand atroom temperature for 10 minutes. Thereafter, to separate the TMAderivatized Fab' fragment from the TMA, the reaction mixture was appliedto a Biogel P-6 column (Biorad Laboratories, Richmond, Calif. 94804) andeluted in citrate buffered saline (50 mM ammonium citrate, 100 mM NaCl,1 mM DTPA, pH 6.3) and eluted into 0.1M NaCl at 1˜DTPA, pH 6-3 50 mMammonium citrate to 0.1 mole NaCl and 1 mM pH 6.3. 2.3 ml fraction ofprotein containing eluate was collected. The A₂₈₀ indicated theconcentration of the desired product to be 0.49 mg/ml or 9.8 μM(assuming no absorbance of TMA). The corresponding maleimideconcentration in the coupled product was determined to be 4.6 μM,resulting in 0.47 TMA molecules per Fab' fragment. The resultant coupledproduct is designated herein as "xCEM-Fab'-TMA."

(c) Coupling xCEM-Fab'-TMA With xCEM-Fab'SH To Form xCEM-TMA-xcEM

The products from (a) and (b) above were combined in model reactions inthe following ratios to test the coupling properties of TMA derivatizedFab'-like fragments. The product of these reactions was designated asxCEM-TMA-xCEM (the "Fab'" having been deleted for clarity):

    ______________________________________                                        xCEM-Fab' -TMA                                                                              xCEM-Fab' SH                                                    .49 mg/ml     (1.3 mg/ml)  Molar Ratio                                        ______________________________________                                        300 μl     115 μl    1:1                                                300 μl      57 μl    2:1                                                150 μl     115 μl    1:2                                                ______________________________________                                    

The yields of the model compound, xCEM-TMA-xCEM, varied as a function ofthe ratio of the reactants:

    ______________________________________                                        CEM-Fab' -TMA/CEM-Fab' SH                                                                         % Yield Of                                                (Molar Ratio)       (CEM-Fab' ).sub.2 TMA                                     ______________________________________                                        2:1                 46%                                                       1:1                 40%                                                       1:2                 19%                                                       ______________________________________                                    

Thus, the best yields of xCEM-TMA-xCEM were obtained when the Fab'-likefragments bearing the maleimide moiety were in excess over the Fab'-likefragments containing the free sulfhydryl group.

Example 12 Preparation Of The Bifunctional Antibody-Like Compound,xCHA-TMA-xCEM, Having Specificity For The Imaging Agent, In-EDTA, andCEA (a) Preparation Of F(ab')₂ Fragments From Intact Antibody

Unless otherwise described herein, the monoclonal antibodies designatedas xCHA, CHA, CEM, ZCE and CYA herein were digested with pepsin toproduce F(ab')₂ fragments according to the following procedure. Antibodysolutions, having an antibody concentration of 5.15 mg/ml as determinedby their absorbance at 280 nm ("A₂₈₀ "), were dialyzed in acetatebuffered saline (0.1M sodium acetate, 0.1M NaCl, pH 4.1) overnight at 4°C. Thereafter, a concentrated pepsin solution was added to the dialyzedsolution containing approximately 2% antibody by mass in pepsin. Thereaction mixture was then incubated from 4.48 hours at 37° C. Thereaction was terminated by addition of NaHCO₃ until the pH was ˜8. TheF(ab')₂ fragments were purified by a variety of techniques, includinggel filtration on a Sephadex® G-150 column (Pharmacia, Piscataway,N.J.); high pressure liquid chromatography, using as a matrix eitherFast Flow S (Pharmacia) or TSK-GEL SP-TOYOPEARL® 650S cation exchangeresin (Tosoh Corp., Japan), and 0.17M sodium acetate pH 4.5 with a NaClgradient as the elution buffer. After isolation, the F(ab')₂ fragmentwas dialyzed in borate buffered saline (50 mM sodium borate, 50 mM NaCl,pH 8.2). The dialyzed solutions containing F(ab')₂ fragments were usedin the subsequent reduction steps.

(b) Reduction of xCHA-F(ab')₂ to xCHA-Fab'SH

To 4 ml of the above dialyzed solution containing 5.7 mg/ml xCHA-F(ab')₂was added 118 mg of NaHCO₃ which adjusted the pH to 8. Thereafter, 8 μlof 0.5M diethylenetriaminepentaacetic acid ("DTPA") was added and thereaction was allowed to proceed at 37° C. for 15 minutes. Upon thefurther addition of 160 μl of 0.5M cysteine, the reaction mixture wasincubated for 10 minutes at 37° C. The reaction mixture was applied to a50 ml P-6 column (Biorad Laboratories, Richmond, Calif. 94804) that waspre-equilibrated and eluted with citrate buffered saline (50 mM ammoniumcitrate, 100 mM NaCl. 1 mM DTPA, pH 6.3). During elution. a 7.84 mlprotein fraction was collected. The absorbance of the fraction at 280 nm("A₂₈₀ ") indicated that the protein concentration of the fraction was55 μM. The sulfhydryl content of the protein fraction was determined tobe 110 μM by adding 5,5'-dithiobis-(2-nitrobenzoic acid) ("DTNB") andborate buffered saline (50 mM sodium borate, 50 mM NaCl, pH 8.2) to analiquot of the reaction mixture and measuring the absorbance differencesat 412 nm between the aliquot containing DTNB and a blank. The moleratio of sulfhydryl groups to Fab' fragment for xCHA-Fab'SH wascalculated to be 2.0:1. The resulting reduced fragment was designatedxCHA-Fab'SH.

(c) Preparation of a F(ab')₂ Fragment From Anti-CEA

A mouse/human chimeric antibody, having specificity for CEA anddesignated as "xCEM," was cloned and expressed according to theprocedure taught in Beidler et al., J. Immunol., 141 pp. 4053-4060(1988). The "xCEM" antibody was digested with pepsin for 3 hoursaccording to the procedure described in Example 11(a) herein to producethe corresponding F(ab')₂ fragment, designated herein as xCEM-F(ab')₂.

(d) Reduction of xCEM-F(ab')₂ to xCEM Fab'SH

To 6.5 ml of the final dialyzed solution from step (c) above, whichcontained 8.46 mg/ml of xCEM-F(ab')₂, was added 13 μl of 0.5Mdiethylenetriaminepentaacetic acid ("DTPA"). The reaction mixture wasincubated for 15 minutes at 37° C. followed by the addition of 260 μl of0.5M cysteine and a further incubation at 37° C. for 10 minutes.Thereafter, the resultant mixture was applied to a 50 ml Biogel P-6column (Biorad Laboratories, Richmond, Calif. 94804) which waspre-equilibrated with citrate buffered saline (50 mM ammonium citrate.100 mM NaCl, 1 mM DTPA, pH 6.3). Upon elution of the column with thissame buffer, a 10.8 ml protein fraction was collected. The absorbance ofthe eluate at 280 nm indicated the protein concentration to be 104 μM.The sulfhydryl content, as determined by the procedure in Example 11(a)herein, was calculated to be 1.6 per fragment. The resulting reducedfragment was designated xCEM-Fab'SH.

(e) TMA derivatization of xCEM-Fab'SH

The reduced fragment xCEM-Fab'SH, was coupled totris(2-N-maleimidoethyl)amine ("TMA") according to the followingprocedure. To 12 mg of TMA dissolved in 200 μl of dimethylformamide(DMF) in a test tube was added 10 ml of citrate buffered saline (50 mMammonium citrate, 100 mM NaCl, 1 mM DTPA, pH 6.3) that was 104 μM inxCEM-Fab'SH. The mixture turned cloudy at first but cleared when theaddition of 10 ml of xCEM-Fab'SH was complete. A white precipitateremained on the bottom and sides of the test tube. The reaction wasallowed to stand at room temperature for 10 minutes. Thereafter, thereaction mixture was applied to a 200 ml P-6 column (BioradLaboratories) that had been pre-equilibrated with citrate bufferedsaline (50 mM ammonium citrate, 100 mM NaCl, 1 mM DTPA, pH 6.3). Uponelution of the column with the same buffer a 23 ml fraction containingxCEM-Fab'-TMA was collected. The absorbance of the fraction at 280 nmindicated that its protein concentration was 2.3 mg/ml or 47 μM.

Using maleimide back titration, the maleimide concentration of theprotein fraction was also determined. Specifically, to 200 μl of the 23ml fraction containing xCEM-Fab'-TMA was added 20 μl of 1.0 mM cysteine.The mixture was allowed to stand at room temperature for 5 minutes.Thereafter 10 μl of 5,5'-dithiobis-(2-nitrobenzoic acid) ("DTNB") and770 μl of borate buffered saline (50 mM sodium borate, 50 mM NaCl, pH8.2) were added. The reaction mixture was spectrophotometricallyanalyzed at 412 nm and the maleimide concentration in the 23 ml fractionwas determined to be 34 μM. The mole ratio of available maleimide perxCEM-TMA molecule was calculated to be 0.72.

(f) conjugation of xCHA-Fab'SH with xcEM-Fab'-TMA

To 7.2 ml (396 nmol) of the 55 μM xCHA-Fab'SH solution from Example12(b) herein was added with stirring 11.6 ml (545 nmol) of the 47 μMxCEM-Fab'-TMA solution from Example 12 (e) above. The ratio ofxCHA-Fab'/maleimide in this reaction mixture was calculated to be 1.0:1.The reaction mixture was allowed to stand at room temperature for 4hours at which time the reaction was stopped by the addition of 20 μl ofa 1M solution of the alkylating agent, N-ethylmaleimide ("NEM"). Theproduct, xCHA-TMA-xCEM, was purified from the reaction mixture by avariety of techniques which included HPLC (high pressure liquidchromatography) and using a Fast Flow S (Pharmacia, Piscataway, N.J.) ora TSK-GEL SP-TOYOPEARL® 650s (Tosoh Corp., Japan) matrix; and gelfiltration on a Sephadex® G-150 column (Pharmacia, Piscataway, N.J.).Those skilled in the art are familiar with protein separation techniquesinvolving HPLC, and gel filtration.

Example 13 Preparation Of The Bifunctional Antibody-Like Compound,ZCE-BMP-CHA, Having Specificity For CEA And In-EDTA (a) Preparation OfThe F(ab')₂ Fragment Of Anti In-EDTA

The antibody to In-EDTA, designated herein as CHA, was digested withpepsin according to the procedure in Example 12(a) to produce itscorresponding F(ab')₂ fragment, CHA-F(ab')₂.

(b) Reduction of CHA-F(ab')₂ to CHA-Fab'SH

A 5 ml aliquot of a final dialyzed solution from step (a) above, whichcontained 10 mg/ml CHA-F(ab')₂. was reduced and purified on a P-6 column(Biorad Laboratories, Richmond, Calif. 94804) according to the procedurein Example 12(b). Upon elution of the P-6 column (Biorad Laboratories),a 12 ml protein fraction was collected. Based upon the absorbance of thefraction at 280 nm, the concentration of the F(ab') fragment was 86 μM.The sulfhydryl concentration of the protein fraction was determined tobe 163 μM using 5,5'-dithiobis-(2-nitrobenzoic acid) ("DTNB"). The molarratio of sulfhydryl groups to Fab' fragments for CHA-Fab'SH wascalculated to be 1.9:1.

(c) BMP Derivatization of CHA-Fab'SH

To 1 ml of a 50:50 solution of DMF/H₂ O was added 13 mg ofN,N'-bis(3maleimidopropionyl)-2-hydroxy-1,3-propanediamine (hereinafter"BMP") which is commercially available from Sigma Chemical Co., St.Louis, Mo. After dissolution of the BMP, 12 ml of the 86 μM proteinfraction from step (b) above, which contained CHA-Fab'SH, was added tothe BMP solution. The reaction mixture was allowed to stand for 10minutes at room temperature. Thereafter, the reaction mixture wasapplied to and eluted from a 200 ml P-6 column (Biorad Laboratories)with citrate buffered saline (50 mM ammonium citrate, 100 mM NaCl, 1 mMDTPA, pH 6.3). A 27 ml protein fraction, which contained theCHA-Fab'BMP, was collected. The A₂₈₀ indicated the CHA-Fab'BMPconcentration to be 33 μM.

The maleimide content of the protein fraction was determined on a 300 μlaliquot of the above fraction using 20 μl of 1 mM cysteine, 10 μl of5,5'-dithiobis-(nitrobenzoic acid) ("DTNB"), and 670 μl of boratebuffered saline pH 8.2, as described in Example 12(e) herein. Themaleimide content of the fraction was determined to be 33 μM. The numberof maleimide moieties available per xCHA-Fab' was calculated as 1.0.

(d) Reduction of ZCE-F(ab')₂ to ZCE-Fab'SH

A commercially available monoclonal antibody to CEA, licensed from JeanPierre Mach, University of Lausanne, Lausanne, Switzerland, anddesignated herein as ZCE, was digested with pepsin according to theprocedure in Example 11(a) to produce its corresponding F(ab')₂fragment, designated as ZCE-F(ab')₂.

Dr. Mach refers to this antibody as Mab35 in his publications.

To a 4 ml aliquot of final solution from the pepsin digestion above,which contained 10 mg/ml ZCE-F(ab')₂, was added 10 μl of 0.5M DTPA andthe reaction mixture was allowed to incubate for 10 minutes at 37° C.Thereafter, 160 μl of 0.5M cysteine was added and the reaction mixturewas incubated at 37° C. for a further 10 minutes. The reaction mixturewas then applied to a 40 ml P-6 column (Biorad Laboratories, Richmond,Calif.) that was pre-equilibrated and eluted with citrate bufferedsaline (50 mM ammonium citrate, 100 mM NaCl, 1 mM DTPA, pH 6.3). A 11.3ml protein fraction was collected, which based upon its absorbance at280 nm (A₂₈₀) was 79 μM in the reduced protein. The sulfhydryl contentof the protein fraction was determined to be 149 μM, using DTNB asdescribed in Example 12(b). The ratio of sulfhydryl groups per ZCE-Fab'fragment was calculated to be 1.9:1. The resultant fragment isdesignated ZCE-Fab'SH.

(e) conjugation of CHA-Fab'BMP to ZCE-Fab'SH

To the 79 μM solution of ZCE-Fab'SH from Example 13(d) above was addeddropwisely an amount of the 33 μM solution of CHA-Fab'BMP sufficient toprovide a 1:1 ratio of ZCE-Fab'SH to reactive maleimide. The reactionmixture was allowed to stand overnight at 4° C. Thereafter, anyunreacted sulfhydryl was blocked by the addition of 16 mg of DTNB to thereaction mixture, thereby providing a concentration of approximately 1mM DTNB.

The resultant bifunctional antibody-like compound, designated asCHA-BMP-ZCE, was purified from the reaction mixture by a variety oftechniques including high pressure liquid chromatography (HPLC) using asmatrices either Fast Flow S (Pharmacia) or TSK-GEL SP-TOYOPEARL® 650scation exchange resin (Tosoh Corp., Japan); and by gel filtration on aSephadex® G-150 column (Pharmacia, Piscataway, N.J.). Those of ordinaryskill in the art are familiar with protein purification via thetechniques of HPLC, and gel filtration.

Example 14 Preparation of Trifunctional Antibody-Like Compounds

Select three different intact antibodies designated herein as Ab₁, Ab₂,and Ab₃, which have the desired specificities and avidities. Theantibodies are individually digested with pepsin according toconventional procedure, such as described in Example 12(a) hereinyielding F(ab')₂ fragments designated as F(ab')₂, F₂ (ab')₂, and F₃(ab')₂, respectively.

The F(ab')₂ fragments derived from each of the three antibodies arereduced with cysteine (or other similar reducing agent) to theirrespective Fab' fragments, i.e., F₁ ab', F₂ ab', and F₃ ab', using aconventional procedure, such as described in Example 12(b) herein.

The three reduced Fab' fragments are selectively coupled together toform a trifunctional antibody-like compound via two trifunctionalcoupling agents of the present invention, e.g.,tris(2-maleoylglycylaminoethyl)amine ("TMG") from Example 8.Procedurally, F₁ ab'-SH, which has been dissolved in an aqueous buffer,pH 5-8 , preferably pH 5-7, is added to a 30-fold molar excess of TMGdissolved in DMF. The reaction mixture is incubated at room temperaturefor 10 minutes. Thereafter, the reaction mixture is applied to a P-6column (Biorad Laboratories, Richmond, Calif.) that has beenpre-equilibrated and which is eluted with citrate buffered saline (50 mMammonium [or sodium] citrate, 100 mM NaCl, 1 mM DTPA, pH 6.3). Theprotein fraction from the P-6 column (Biorad Laboratories) contains thepurified Fab'-TMG which contains at least one maleimide moiety capableof coupling to a second reduced Fab' fragment.

To accomplish a second coupling to the TMG, the protein fractioncontaining the purified F₁ ab'TMG is added dropwise to the secondreduced Fab' fragment, F₂ ab'SH which is in the same citrate buffer asused to elute the P-6 column. The second coupling reaction is allowed toproceed for 3 hours at room temperature. Thereafter, the extrasulfhydryls on the F₂ ab'SH moiety of the coupled product. F₁ ab'-TMG-F₂ab'SH, are protected by a reversible protecting agent, such as DTNB(55'-dithio-bis-(2-nitrobenzoic acid). Protection is begun by addingsufficient DTNB to achieve a final concentration of approximately 1 mMin the citrate buffered saline (50 mM ammonium citrate, 100 mM NaCl, 1mM DTPA, pH 6.3) that contains the F₁ ab'-TMG-F₂ ab'SH. Thereafter, thereaction mixture is incubated for 10 minutes at room temperature. Theresulting protected intermediate, Fab'-TMG-F₂ ab'-S-blocking agent, ispurified by high pressure liquid chromatography (HPLC), using a matrixcomprising either Fast Flow S (Pharmacia, Piscataway, N.J.) or TSK-GELSP-TOYOPEARL® 650s (Tosoh Corp., Japan); or by preparative gelfiltration, such as on a column containing Sephadex® G-150 brandsuperfine resin (Pharmacia, Piscataway, N.J.).

Once purified, the protected intermediate, is deblocked in boratebuffered saline (50 mM sodium borate, 50 mM NaCl, pH 8.2) to which isadded a molar excess of 1 mM cysteine, DTT (dithiothreitol), or asimilar reducing agent. The deblocked and reduced bifunctionalintermediate is further purified by applying the reaction mixture to aP-6 column (Biorad Laboratories) which has been pre-equilibrated withand which is eluted with the just described citrate buffered saline, pH6.3. The reduced bifunctional intermediate, F₁ ab'-TMG-F₂ ab'-SH, is nowready for coupling to a third maleimide derivatized Fab' fragment.

The third Fab' fragment is derivatized by being added to a 30-fold molarexcess of TMG (or other trivalent coupling agent of the presentinvention) as described for the first Fab'-like fragment mentioned inthis example. The reaction mixture is incubated at room temperature for10 minutes. Thereafter, the F₃ ab'-TMG in the reaction mixture ispurified by applying the reaction mixture to a P-6 column (BioradLaboratories) that has been pre-equilibrated with and which is elutedwith the described citrate buffered saline, pH 6.3.

Final coupling to produce the trivalent antibody-like compound of thepresent invention is accomplished by adding F₃ ab'-TMG to a solution ofthe above described citrate buffered saline (pH 6.3) containing the F₁ab'-TMG-F₂ ab'SH and then allowing the reaction to proceed for 3 hoursat room temperature. Thereafter, the reaction is stopped, such as by theaddition of the alkylating agent, N-ethylmaleimide, to the reactionmixture. Purification of the trivalent antibody-like compound(Fab'-TMG-F₂ ab'-TMG-F₃ ab') is accomplished by HPLC, or preparative gelfiltration (e.g. Pharmacia's Sephadex® brand G-150 superfine resin),which are techniques well known to those of ordinary skill in the art.

Example 15 Preparation Of The Trifunctional Antibody-Like Compound:CHA-BMP-GYA-BMP-ZCE

Three different antibodies, which were designated as CHA, CYA and ZCE,were the source of the Fab' fragments that were coupled by two trivalentcoupling agents to form the trifunctional antibody-like compound,CHA-BMP-CYA-BMP-ZCE. In this example, the monoclonal antibody designatedas CHA had specificity for the chelate complex In-EDTA or In-chelated byderivatives of EDTA, such as EOTUBE.

The monoclonal antibody designated as "CYA" had specificity for thechelate complex "Y-DTPA" and the monoclonal antibody designated as "ZCE"had specificity for carcinoembryonic antigen ("CEA").

(a) Preparation of F(ab')₂ Fragments

The F(ab')₂ fragments of CHA, CYA, and ZCE, which are designated asCHA-F(ab')₂, CYA-F(ab')₂, and ZCE-F(ab')₂ respectively, were prepared byindividually digesting the respective antibody with pepsin according tothe procedure described in Example 12(a).

(b) Reduction of CYA-F(ab')₂ to CYA-Fab'SH

To 1.0 ml of borate buffered saline (50 mM sodium borate, 50 mM NaCl, pH8.2) containing 8.1 mg/ml of CYA-F(ab')₂, was added 2 μl of 0.5Mdiethylene-triaminepentaacetic acid (DTPA) and 40 μl of 0.5M cysteine.The reaction mixture was allowed to proceed for 10 minutes at 37° C.Thereafter, the reaction mixture was applied to a 15 ml P-6 column(Biorad Laboratories, Richmond, Calif. 94804) that had beenpre-equilibrated and eluted with citrate buffered saline (50 mM ammoniumcitrate, 100 mM NaCl, 1 mM DTPA, pH 6.3). A 3.0 ml protein containingfraction was collected from the column, which based upon its absorbanceat 280 nm (A₂₈₀) had a protein (reduced Fab') concentration of 48 μM.The concentration of free sulfhydryl groups in the protein fraction wasdetermined to be 118 μM, using the procedure in Example 12(b) herein.Thereafter, the ratio of free sulfhydryl groups per Fab' fragment wascalculated to be 2.5:1.

(c) Preparation of CHA-BMP-ZCE-SH

The bifunctional antibody-like compound, CHA-BMP-ZCE, having a blockedsulfhydryl, was prepared according to the procedure of Examples13(a)-(e). Thereafter, to a 2.5 ml aliquot containing 3.9 mg/ml of thepurified blocked CHA-BMP-ZCE in borate buffered saline (50 mM sodiumborate 50 mM NaCl, pH 8.2) was added 5 μl of 0.5Mdiethylenetriaminepentaacetic acid ("DTPA"). The reaction mixture wasincubated for 15 minutes at 37° C. followed by the subsequent additionof 100 μl of 0.5M cysteine. The reaction mixture was further incubatedfor 10 minutes at 37° C., which effected deblocking. Thereafter, thereaction mixture was applied to a Biogel P-6 column (BioradLaboratories) that had been pre-equilibrated and which was eluted withcitrate buffered saline (50 mM ammonium citrate, 100 mM NaCl, 1 mM DTPA,pH 6.3). A 5.8 ml protein fraction was collected, which based upon itsabsorbance at 280 nm (A₂₈₀), had a 15 μM protein (reduced bifunctionalantibody) concentration. The sulfhydryl concentration of the proteinfraction was subsequently determined, according to the procedure inExample 10(a).

(d) Coupling CYA-BMP with CHA-BMP-ZCE-SH to form CHA-BMP-ZCE-BMP-CYA

To 5.5 ml of the citrate buffered saline solution that was 15 μM inCHA-BMP-ZCE-SH (from step (c) above) was added an equimolar amount ofCYA-BMP similarly dissolved in citrate buffered saline (50 mM sodiumcitrate, 100 mM NaCl, pH 6.3). The reaction was allowed to proceed for 3hours at room temperature and then was terminated by the addition ofN-ethylmaleimide as described in Example 14 herein. The resultanttrifunctional antibody-like compound, designated as CHA-BMP-ZCE-BMP-CYA,was purified by gel filtration on Sephadex® G-150 (Pharmacia, Piscatway,N.J.), eluting with borate buffered saline (50 mM sodium borate, 50 mMNaCl, pH 8.2). Fractions 38.43 were collected and pooled to yield 3.2 mlof a solution containing 0.66 mg/ml of the purified product. A 2.0 mlaliquot of the pooled fractions was then dialyzed overnight in 0.17Msodium acetate, pH 4.5, for subsequent HPLC purification on a Mono Smatrix (Pharmacia).

The uncorrected binding capacity for the In-EOTUBE complex by theCHA-BMP-ZCE-BMP-CYA in the 0.66 mg/ml pooled fraction was determined tobe 76% of theoretical capacity. The control for the same run exhibited abinding capacity of 4%. The Y-MeTUBD binding capability was 82% of itstheoretical value.

Example 16 Synthesis Of The Trivalent Antibody-Like Compound:xCEM-TMG-xCHA-TMG-xCEM (a) Digestion of "xCHA" and "xCEM" toxCEM-F(ab')₂ and xCEM-F(ab')₂ Respectively.

Intact chimeric monoclonal antibody to the In-EDTA complex is designatedherein as "xGHA." Intact chimeric monoclonal antibody to CEA isdesignated herein as "xCEM." The preparation of these antibodies was asreferenced in Examples 11 and 12 herein. Intact xCHA and xCEM antibodieswere individually digested to their respective F(ab')₂ fragments byincubating each with 3% (pepsin:antibody) at 37° C. for 5 hours inacetate buffered saline (100 mM sodium acetate, 100 mM sodium chloride,pH 4.1). The digests were terminated by neutralization of the pH.Thereafter the digests were dialyzed in borate buffered saline (50 mMsodium borate 100 mM sodium chloride, pH 8.2) to provide thecorresponding F(ab')₂ fragments designated as xCHA-F(ab')₂ andxCEM-F(ab')₂ respectively.

(b) Reduction of xCEM-F(ab')₂ to xCEM-Fab'SH

To 6 ml of xCEM-F(ab')₂ (17 mg/ml) obtained from Step (a) above wasadded 2.0 ml of borate buffered saline (50 mM sodium borate 100 mMsodium chloride, pH 8.2) and 16 μl of 0.5M diethylenetriaminepentaaceticacid ("DTPA") to reach a final DTPA concentration of 1 mM. The reactionmixture was incubated at 37° C. for 10 minutes, followed by the additionof 360 μl of 0.5M cysteine, and a further incubation for 10 minutes at37° C. The cysteine was removed by gel filtration on a 2.5×19 cm P-6 DGcolumn (Biorad Laboratories, Richmond, Calif. 94804) that had beenpre-equilibrated with and which was eluted with citrate buffered saline(50 mM ammonium citrate, 100 mM NaCl, 1 mM DTPA, pH 6.3). Upon elution,a 19.6 ml protein fraction was collected, which based upon itsabsorbance at 280 nm (A₂₈₀) was 90 μM in the reduced Fab'fragment--xCEM-Fab'SH. The free sulfhydryl concentration of the proteinfraction was determined by reaction with DTNB (as per Example 10(a)) tobe 159 μM. The ratio of free sulfhydryl per reduced Fab' fragment wascalculated to be 1.8:1.

(c) Derivatization of xCEM-Fab'SH with TMG

The reduced Fab' fragment, xCEM-Fab'SH, was derivatized with a 30 foldmolar excess of tris[2-N-(maleoylglycyl)aminoethyl]amine("TMG"). Inparticular, 19.5 ml of xCEM-Fab'SH (1.8 μmoles) in citrate bufferedsaline from step (b) above was added with stirring to 176 μl of 314 mMTMG (53 μmoles) in DMF. After 10 minutes at 23° C., excess TMG wasremoved on a 2.5×45 cm P-6 DG column (Biorad Laboratories) that waspre-equilibrated and eluted with citrate buffered saline (50 mM ammoniumcitrate. 100 mM NaCl, 1 mM DTPA, pH 6.3). A 26.8 ml protein fraction wascollected, which based upon its absorbance at 280 nm (A₂₈₀) contained3.3 mg/ml or was 67 μM in the derivatized fragment--xCEM-Fab'-TMG. Thedetermination of active maleimides by cysteine back titration (perExample 12(e)) indicated 1.16 active maleimides per Fab' fragment.

(d) Reduction of xCHA-F(ab')₂ To xCHA-Fab'SH

A 2.7 ml aliquot of xCHA-F(ab')₂ (9.2 mg/ml) was incubated with 1 mMDTPA for 10 minutes at 37° C. To this reaction mixture was then added 6μl of 0.5M dithiothreitol ("DTT") and the reaction mixture was furtherincubated at 37° C. for a further 10 minutes. The DTT was removed by gelfiltration on a 1.5×25 cm P-6 DG column (Biorad Laboratories) that waspre-equilibrated and eluted with citrate buffered saline (50 mM ammoniumcitrate, 100 mM NaCl, 1 mM DTPA, pH 6.3). A 7.6 ml protein fraction wascollected, which based upon its absorbance at 280 nm (A₂₈₀) was 59 μM inthe desired xCHA-Fab'SH. The free sulfhydryl concentration of thefraction was determined to be 236 μM. The ratio of free sulfhydrylgroups per reduced Fab' fragment was calculated to be 4.7:1.

(e) Coupling xCHA-Fab'SH and xCEM-Fab'TMG

To 7.5 ml of xCHA-Fab'SH from step (d) above was added 26.2 ml ofxCEM-Fab'-TMG from Step 15(c) above and the reaction mixture wasincubated at 23° C. for 100 minutes. The reaction was terminated by theaddition of 34 μl of 1M N-ethylmaleimide, an alkylating agent Thereaction mixture was then concentrated to 12 ml by ultrafiltration. Theconcentrated reaction mixture was purified by gel filtration on a 2.6×96cm G-150 superfine column (Pharmacia, Piscataway, N.J.), having a bedvolume of 500 ml. The flow rate was approximately 0.2 ml/min. and 5 mlfractions were collected. The A₂₈₀ trace of the elution patternindicated 3 major products. The desired trivalent antibody likecompound, designated herein as xCEM-TMG-xCHA-TMG-xCEM, was found as themiddle product, i.e., in fractions 38-41. The identity of thexCEM-TMG-xCHA-TMG-xCEM was confirmed by high pressure liquidchromatography (HPLC) gel filtration and by sodium dodecylsulfate--polyacrylamide gel electrophoresis (SDS-PAGE) using a 7.5%acrylamide gel.

What is claimed is:
 1. A compound of the formula: ##STR22## wherein X is##STR23## wherein k=1 or 0; wherein Z is ##STR24## wherein s=1 or 0;wherein n=1;wherein q=1 or 0; wherein Y is ##STR25## wherein Y' is##STR26## wherein p or m may be the same or different and are integersranging from 0 to 20 with the provisos that when n=1, p and m are eachan integer that is at least 1 and the sum of p and m is an integerranging from 2 to 20; wherein R¹ is straight or branched chain loweralkyl having from 1 to 6 carbon atoms or lower alkoxy having from 1-6carbon atoms; and wherein R² is hydrogen phenyl, --COOH, or straight orbranched chain lower alkyl having from 1-6 carbon atoms optionallymonosubstituted by --NH₂, --OH, or --COOH.
 2. The compound according toclaim 1 wherein Y is ##STR27##
 3. The compound according to claim 2which is: ##STR28##
 4. The compound according to claim 2 which is:##STR29##
 5. A compound according to claim 1 which is: ##STR30##
 6. Thecompound according to claim 1 which is: ##STR31##
 7. The compoundaccording to claim 1 wherein n=1, s=0, and m and p are each an integerthat is at least 1 and the sum of m and p is an integer from 2 to about20.
 8. The compound having the formula: ##STR32##
 9. The compoundaccording to claim 1 which is: ##STR33##
 10. The compound having theformula: ##STR34##
 11. The compound according to claim 1 which is:##STR35##